Molecular characterization and secreted production of basidiomycetous cell-bound β-glycosidases applicable to production of galactooligosaccharides.

Cell-bound β-glycosidases of basidiomycetous yeasts show promise as biocatalysts in galactooligosaccharide (GOS) production. Using degenerated primers designed from Hamamotoa singularis (Hs) bglA gene, we newly identified three genes that encode cell-bound β-glycosidase from Sirobasidium magnum (Sm), Rhodotorula minuta (Rm), and Sterigmatomyces elviae (Se). These three genes, also named bglA, encoded family 1 glycosyl hydrolases with molecular masses of 67‒77 kDa. The BglA enzymes were approximately 44% identical to the Hs-BglA enzyme and possessed a unique domain at the N-terminus comprising 110 or 210 amino acids. The Sm-, Rm-, and Se-BglA enzymes as well as the Hs-BglA enzyme were successfully produced by recombinant Aspergillus oryzae, and all enzymes were entirely secreted to the supernatants. Furthermore, addition of some nonionic detergents (e.g. 0.4% [v/v] Triton-X) increased the production, especially of the Hs- or Se-BglA enzyme. Out of the BglA enzymes, the Se-BglA enzyme showed remarkable thermostability (∼70°C). Additionally, the Sm- and Se-BglA enzymes had better GOS yields, so there was less residual lactose than in others. Accordingly, the basidiomycetous BglA enzymes produced by recombinant A. oryzae would be applicable to GOS production, and the Se-BglA enzyme appeared to be the most promising enzyme for industrial uses.